生淀粉水解α-淀粉酶AmyZ1热稳定性提升的分子改造

Molecular modification of raw starch hydrolase AmyZ1 in the enhancement of thermal stability

生淀粉水解α-淀粉酶是一种具有生淀粉水解能力的α-淀粉酶,能够在淀粉糊化温度以下降解生淀粉颗粒,适用于淀粉冷水解。ΑmyZ1是来源于海洋微生物的生淀粉水解酶,其催化底物转化应用时热稳定性不足。与热稳定α-淀粉酶结构进行比对分析,发现AmyZ1在结构域B区域多了一个柔性loop区。通过同源建模,确定突变位点,构建缺失柔性区的突变酶AmyZ1∶ΔTG,并对突变酶的酶学性质、Ca^(2+)依赖以及水解高浓度玉米生淀粉的能力进行分析。AmyZ1∶ΔTG最适作用温度为45℃,在35℃~55℃范围内酶活力维持在60%以上。在30℃和35℃条件下,半衰期可分别达到18 h和15 h,是出发酶AmyZ1的10倍和15倍。突变酶AmyZ1∶ΔTG蛋白结构的柔性降低,导致AmyZ1∶ΔTG的催化效率有所降低。与出发酶AmyZ1相比,AmyZ1∶ΔTG的催化性能受外源Ca^(2+)的影响较小,酶活力对外源Ca^(2+)的依赖有所降低,但Ca^(2+)有助于提升其热稳定性。此外,在35℃的作用条件下,AmyZ1∶ΔTG对300 g/L玉米生淀粉的水解率为40%,接近于出发酶AmyZ1。

Raw starch degrading enzymes areα-amylases with the ability to hydrolyze raw starch.It can degrade raw starch granules below the starch pasting temperature and suitable for cold hydrolysis of starch.The raw starch hydrolase AmyZ1 is derived from a marine microorganism and is not thermally stable.The mock structure of AmyZ1 was obtained by homology modeling.AmyZ1∶ΔTG is a mutant enzyme with deletion of the flexible region was constructed by sentinel mutation.The catalytic property,Ca^(2+)dependence,and ability to hydrolyze high concentration raw maize starch of the mutant were evaluated.The optimum temperature of AmyZ1∶ΔTG was 45℃,and the enzyme activity was maintained above 60% in the range of 35℃-55℃.The thermal stability of AmyZ1∶ΔTG was improved and the half-lives of 30℃ and 35℃ reached 18 h and 15 h,which were 10 and 15 times of AmyZ1,respectively.The decreased flexibility of the protein resulted in a decrease in the catalytic efficiency of AmyZ1∶ΔTG.Exogenous Ca^(2+)has less effect on the catalytic performance of AmyZ1∶ΔTG,but it can improve the thermal stability of AmyZ1∶ΔTG,compared to that of AmyZ1.Using 300 g/L corn raw starch as the substrate,the hydrolysis rate of AmyZ1∶ΔTG was 40%,which was similar to AmyZ1.